In the past few years, conjugated polymer nanoparticles (CPNs) have been successfully prepared and applied in the biological field because of their unique opto-electronic properties. The rapid development of CPNs is mainly attributed to their simple synthesis procedures and easy separation steps. The advantages of CPNs include high brightness, excellent photostability, low cytotoxicity, high quantum yield and versatile surface modification. The functionalization of CPNs with specific recognition elements imparts them good ability for targeted recognition and imaging in vitro and in vivo. CPNs can be applied to deliver drug and gene, and simultaneously to real-time monitor the release process due to their self-luminous characteristics. Moreover, CPNs can sensitize oxygen molecules to generate reactive oxygen species (ROS) which can kill adjacent bacteria and tumor cells. In this tutorial review, we provide a recent development of the preparation methods, properties, and functionalization strategies of CPNs, especially discussing their biological applications in targeted imaging, drug/gene delivery and biomedicine. The challenges and outlooks in this field will also be discussed.